Functional properties of the uptake of amines in immortalised peptidergic neurones (transport‐P)

Abstract

1Most neurotransmitters are inactivated by uptake into presynaptic nerve terminals and into glial cells. We recently provided evidence for uptake of amines in postsynaptic neurones. Uptake was evident at nanomolar concentrations of prazosin, but at concentrations of unlabelled prazosin greater than 10-M, there was a further activation of uptake, manifested by a paradoxical increase in accumulation of the radioligand. We have now studied further characteristics of amine uptake in immortalised gonadotrophin-releasing hormone (GnRH) neurones. Control cells included SK-N-SH neuroblastoma cells (which possess presynaptic type amine transporters) and non-neuronal (COS-7) cells. 2 [3H]-prazosin bound to intact GnRH cells and was displaced by unlabelled prazosin in concentrations of 10-to 10-' M. However, at higher concentrations of unlabelled prazosin, there was an increase in apparent [3H]-prazosin binding, as we had previously described. This paradoxical increase in accumulation of the radioligand was abolished by desipramine. 3 Desipramine had no effect on the association of prazosin with COS-7 cells. There was no paradoxical increase in accumulation of [3H]-prazosin in COS-7 cells, indicating that this effect requires the presence of a desipramine-blockable uptake process. 4 The increase in binding of the radioligand that was observed in the GnRH cells is not a general property of neuronal transporters; in SK-N-SH cells, there was no increase in accumulation of (-)-[3H]-noradrenaline in the presence of concentrations of unlabelled (-)-noradrenaline greater than 10-7 M. 5 The uptake of prazosin and the increase in accumulation of [3H]-prazosin were abolished in the cold, indicating that this is an active, energy-requiring process. 6 Desipramine-sensitive uptake of prazosin was demonstrable in the GnRH cells in the absence of sodium. Further, the Na+/K+-ATPase inhibitor, vanadate, abolished noradrenaline uptake in SK-N-SH cells but had no effect on prazosin uptake in GnRH cells. Thus, the uptake of prazosin does not derive its energy from the sodium pump.7 Prazosin uptake was inhibited by the V-ATPase inhibitor bafilomycin Al, the H+/Na+ ionophore, monensin and the organic base, chloroquine, indicating that uptake derives its energy from a proton pump. In contrast to other proton-dependent amine transporters, the uptake of prazosin was unaffected by reserpine. 8 Increasing extracellular pH did not increase the uptake of prazosin into GnRH cells, indicating that it is unlikely to be due to non-specific diffusion and concentration of a lysosomotropic drug into intracellular acidic particles. 9 The uptake of prazosin was unaffected by steroid hormones.10 In COS-7 cells transfected with ocl-adrenoceptor cDNA, [3H]-prazosin was displaced by unlabelled prazosin without causing an increase in binding of the radioligand. This indicated that the increase in accumulation of the radioligand is unlikely to be due simply to some function of a,-adrenoceptors.11 Thus, peptidergic neurones possess an uptake process with properties ...

Binding and competitive inhibition of amine uptake at postsynaptic neurones (transport‐P) by tricyclic antidepressants

“…[3H]-prazosin is completely displaced from xl-adrenoceptors (Al-Damluji & Kopin, 1996). The action of imipramine therefore represents inhibition of uptake, rather than inhibition of binding to xl-adrenoceptors.…”

“…While it resembles the presynaptic plasma membrane transporters in that it is blocked by desipramine, it differs from these transporters by its independence of sodium and reliance on protons for a source of energy. Uptake of prazosin differs from the vesicular transporters by its insensitivity to reserpine and blockade by antidepressants (Al-Damluji & Kopin, 1996). Thus, this carrier is distinguishable from other neuronal transporters both by its anatomical location and by its functional properties.…”

“…Primary hypothalamic cell cultures and a cell line of hypothalamic neurones were found to accumulate amines by a desipramine-blockable process (Al-Damluji & Krsmanovic, 1992;Al-Damluji et al, 1993). These cells accumulate amines which are present in nanomolar concentrations in the extracellular space by an energy-dependent process that is linked to a vacuolar-type ATPase (V-ATPase) and requires an electrochemical gradient of protons for its source of energy (Al-Damluji & Kopin, 1996 ). Uptake is due to a carrier which is activated by increasing concentrations of prazosin, resulting in a paradoxical increase in the apparent binding of [3H]-prazosin.…”

The Medicinal Chemistry at the “Polo de Xistoquímica” of the IQ‐UFRJ

“…Este mesmo fenômeno foi observado com os antidepressivos atípicos, como a mianserina e a fluvoxamina. 12 Outros estudos demonstraram que este processo de recaptação de prazosina é dependente da integridade da célula, não ocorrendo em preparações de membrana dos neurônios peptidérgicos estudados. O Transporte P também não foi observado em neurônios pré-sinápticos noradrenérgicos ou em células da glia, demosntrando ser um processo específico dos receptores pós-sinápticos.…”

Neurotransmitter transporters: fruitful targets for CNS drug discovery

“…20 It is possible that antidepressant drugs have other targets in addition to their actions at cell surface monoamine transporters. Al-Damluji and Kopin 22 have described a novel amine uptake process in peptidecontaining hypothalamic neurons, which they named 'transport-P'. Like the vesicular transporters this process is driven by a proton gradient, but it is distinct from the vesicular transporters in being insensitive to reserpine, but sensitive to a variety of tricyclic antidepressants at micromolar concentrations.…”